Molecular mechanisms of vascular calcification and their connection to coronary disease risk

血管钙化的分子机制及其与冠心病风险的关系

基本信息

批准号：
10673742
负责人：
THOMAS QUERTERMOUS
金额：
$ 58.92万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-01 至 2026-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10673742
关键词：
ATAC-seq Address Anatomy Animals ApoE knockout mouse Apolipoprotein E Aryl Hydrocarbon Receptor Atherosclerosis Binding Blood Vessels Cardiovascular Diseases Cell Lineage Cell model Cells Cellular Assay Cellular Morphology Chromatin Chromosome Mapping Complex Coronary Arteriosclerosis Coronary artery Coronary heart disease Development Developmental Process Disease Enhancers Epigenetic Process Fibroblasts Functional disorder Gene Expression Genes Genetic Risk Genetic Transcription Goals Human Human Genome In Vitro Investigation Knock-out Knockout Mice Lesion Link MADH3 gene Maps Medial Mediating Modeling Molecular Morphology Mus Osteogenesis Pathway interactions Phenotype Process Regulation Research Risk Assessment Role Scientist Signal Pathway Signal Transduction Signaling Molecule Smooth Muscle Myocytes Subcellular Anatomy TGFB1 gene Therapeutic Tissues Transposase Vascular Diseases Vascular Smooth Muscle Vascular calcification Wild Type Mouse Work blocking factor calcification cell type disorder risk genome wide association study genome-wide in vivo mouse model novel programs public health relevance recruit single-cell RNA sequencing transcription factor transcription factor USF transcriptome sequencing

项目摘要

The combination of lineage tracing and single cell RNA sequencing (scRNAseq) in mouse atherosclerosis models has created a paradigm shift in our understanding of vascular disease, showing that lesion smooth muscle cells (SMC) undergo phenotypic transitions into derivative cells with multiple complex phenotypes. We identified TCF21 as a coronary artery disease (CAD) associated gene mapped by genome-wide association studies (GWAS) and showed that this gene regulates a disease-related transition of SMC to a fibroblast like phenotype, producing cells we term “fibromyocytes.” Further, we and others have shown that medial SMC can also transition to a second SMC-derived cellular phenotype, characterized by expression of genes known for their role in endochondral bone formation, substantiating and expanding previous work investigating this process that is linked to intimal vascular calcification. We showed that this chondrogenic process, which gives rise to cells we term “chondromyocytes” (CMC), is actively inhibited by two CAD associated genes, one encoding the TGFB1 signaling molecule SMAD3, and the other encoding the environmental sensing aryl hydrocarbon receptor (AHR). Knockout (KO) of both genes in mouse models showed increased transition to CMC, larger lesion size and increased vascular calcification. These studies identified SOX9 as a primary driver of the phenotypic transition to the CMC phenotype. Our longterm goal is to elucidate the molecular mechanisms that mediate the detrimental CMC transition. Our Central Hypothesis postulates that SOX9 is a key initiator of this chondrogenic process in the vascular wall, as it is in endochondral bone formation, and regulation of its expression and function in SMC is intimately linked to vascular calcification and disease risk. Our objective is thus to determine the upstream epigenetic signals that modulate SOX9 expression, and how SOX9 expression contributes to CMC development and vascular calcification. Specifically, in Aim 1 we will employ Sox9 KO and SMC lineage tracing in the ApoE KO mouse atherosclerosis model to characterize the effect of this gene on SMC cell state transitions, and the impact of perturbing these transitions on disease morphology and cellular anatomy. In Aim 2, we will conduct scRNAseq in these mice to characterize the SMC gene expression program downstream of Sox9 in this cell type. Single cell assay of transposase accessible chromatin sequencing (scATACseq) in the same animals will map enhancers genome-wide that are differentially regulated in CMC phenotypic transition, and identify specific transcription factors (TFs) that bind these enhancers to regulate expression of CMC genes. Studies proposed in Aim 3 will employ in vitro studies to characterize the transcriptional and epigenetic mechanism by which SOX9 interacts with the inhibitory factors SMAD3 and AHR, and novel TFs that promote transition to the CMC phenotype. The proposed studies will identify cellular and molecular mechanisms that mediate SMC transition to CMC, and the relationship of this process to vascular calcification and disease risk.

小鼠动脉粥样硬化中谱系跟踪和单细胞RNA测序（SCRNASEQ）的组合模型已经在我们对血管疾病的理解中产生了范式转变，表明病变光滑肌肉细胞（SMC）经历了具有多种复杂表型的衍生细胞的表型过渡。我们将TCF21鉴定为冠状动脉疾病（CAD）与全基因组关联映射的相关基因研究（GWAS），并表明该基因调节SMC与疾病相关的成纤维细胞过渡表型，产生细胞，我们称为“纤维肌细胞”。此外，我们和其他人表明内侧SMC可以也过渡到第二个SMC衍生的细胞表型，其特征在于表达以它们在内向骨骨形成中的作用，证实和扩大了先前的研究与内膜血管钙化有关的过程。我们证明了这种软骨生成过程，这给出了我们称为“软骨细胞”（CMC）的细胞，被两个CAD相关的基因积极抑制，一个编码TGFB1信号分子SMAD3，另一个编码环境感应芳基碳氢化合物受体（AHR）。小鼠模型中两个基因的基因敲除（KO），显示了过渡到 CMC，较大的病变大小和增加的血管钙化。这些研究将SOX9确定为主要驱动力表型过渡到CMC表型。我们的长期目标是阐明分子介导有害的CMC过渡的机制。我们的中心假设假设Sox9是在血管壁中，该软骨生成过程的关键引发剂，就像内侧软骨骨中 SMC中其表达和功能的形成和调节与血管密切相关钙化和疾病风险。因此，我们的目标是确定上游的表观遗传信号调节SOX9表达，以及Sox9表达如何有助于CMC发育和血管钙化。具体而言，在AIM 1中，我们将在ApoE KO鼠标中使用Sox9 KO和SMC谱系跟踪动脉粥样硬化模型以表征该基因对SMC细胞状态过渡的影响以及在疾病形态和细胞解剖结构上扰动这些转变。在AIM 2中，我们将进行Scrnaseq 在这些小鼠中，以这种细胞类型的SOX9下游表征SMC基因表达程序。单身的同一动物中的转座酶可访问染色质测序（SCATACSEQ）的细胞测定将映射在CMC表型过渡中受不同调节的全基因组的增强剂，并确定特定结合这些增强子以调节CMC基因表达的转录因子（TF）。研究提出在AIM 3中，将采用体外研究来表征转录和表观遗传机制 Sox9与抑制因子SMAD3和AHR相互作用，以及促进过渡到CMC的新型TFS 表型。拟议的研究将确定介导SMC转变的细胞和分子机制 CMC以及该过程与血管计算和疾病风险的关系。